Francisella tularensis is a highly infectious, risk-group A select bacterial agent that has the potential to be weaponized. It is the cause of tularemia. The applicants' long-term goal is to understand the pathways of genes and gene products that drive unique virulence and transmissibility traits of the four subspecies of F. tularensis. The objective of this R21 application is to identify genes that are responsible for virulence and transmissibility of the most virulent subspecies of this bacterium. The central hypothesis is that increased virulence and transmissibility are attributable to genetic differences. This hypothesis will be tested by pursuing three specific aims: 1) Design and characterize a reference strain collection representing both F. tularensis subsp. tularensis and supsp. holarctica with temporal and spatial representation; 2) Fabricate representative reference microarrays of the tularensis and holarctica subsp. and systematically probe genome diversity of the strain collection using arrays; and 3) Use paired-end sequence mapping to complement data from the microarray-based studies. The principal approaches and methods to be used include alternative strategies and tactics to systematically catalogue subspecies-specific genome segments. This will be accomplished using DNA microarray analyses (arrays derived from shotgun libraries prepared from the representative subsp. tularensis and holarctica) and a novel algorithm for data sorting followed by DNA sequence analyses and confirmation by Southern blotting and PCR. Second, a narrow (10Kb)-size library from a representative holarctica strain will be used to generate paired-end sequence reads, after which the list will be mapped to the subsp. tularensis genome sequence. This will allow length differences that are subspecies-specific to be identified. The outcomes are collectively significant, because they are expected to provide strong preliminary evidence that strain differences in virulence and transmissibility are attributable to genomic differences. Such data are expected to become the foundation for definitive R01-level investigations involving the functional analyses of candidate genes. These studies will significantly increase our understanding of the pathobiology of this organism, provide candidate gene products for vaccine and therapeutics, and provide loci for development of improved diagnostic and forensic markers.